Directed evolution of polypropylene and polystyrene binding peptides

Abstract

Surface functionalization of biological inert polymers (e.g., polypropylene PP; polystyrene PS) with material binding peptides facilitates an efficient immobilization of enzymes, bioactive peptides or antigens at ambient temperature in water. The developed robust directed evolution protocol enables to tailor polymer binding anchor peptides (PBPs) for efficient binding under application conditions. Key for a successful directed evolution campaign was to develop an epPCR protocol with a very high mutation frequency (60 mutations/kb) to ensure sufficient diversity in PBPs (47 aas LCI: “liquid chromatography peak I”; 44 aas TA2: “Tachystatin A2”). LCI and TA2 were genetically fused to the reporter egfp to quantify peptide binding on PP and PS by fluorescence analysis. The Peptide‐Polymer evolution protocol (PePevo protocol) was validated in two directed evolution campaigns for two PBPs and polymers (LCI: PP; TA2: PS). Surfactants were used as selection pressure for improved PBP binders (non‐ionic surfactant Triton X‐100; 1 mM for LCI‐PP // anionic surfactant LAS; 0.5 mM for TA2‐PS). PePevo yielded an up to three fold improved PP‐binder (LCI‐M1‐PP: I24T, Y29H, E42 K and LCI‐M2‐PP: D31V, E42G) and an up to six fold stronger PS‐binder (TA2‐M1‐PS: R3S, L6P, V12 K, S15P, C29R, R30L, F33S, Y44H and TA2‐M2‐PS: F9C, C24S, G26D, S31G, C41S, Y44Q).

Matter‐tag: A universal immobilization platform for enzymes on polymers, metals, and silicon‐based materials

“…The dissociation constant ( K D ) of YmPh‐LCI was determined to be 2.9·10 −8 M and is comparable to the reported K D values of anti‐His‐tag antibodies (3D5 antibody: 3.4·10 −7 M, PentaHis antibody: 1.0·10 −9 M; Müller, Arndt, Bauer, & Plückthun, ) and polyhistidine‐tags for Ni 2+ ‐NTA (free 6His‐peptide: 1.4·10 −8 M; Knecht, Ricklin, Eberle, & Ernst, ). The K D values can be further reduced to promote stronger adhesion of the Matter‐ tags by applying directed evolution methodologies for instance PeP evo protocol (Rübsam, Weber et al, ) within a KnowVolution campaign (Rübsam, Davari et al, ).…”

“…Besides natural substrates, the adhesion of CBMs to synthetic polymers, for example, polyethylene terephthalate (PET), was reported (Ribitsch et al, 2013;Zhang, Wang, Chen, & Wu, 2013). Further, adhesion-promoting peptides are reported for numerous materials, for instance, for gold (e.g., Midas-2: TGTSVLIATPYV; Kim et al, 2010;GBP1: MHGKTQATSGTIQS; Brown, 1997;Tamerler, Oren, Duman, Venkatasubramanian, & Sarikaya, 2006;AuBP1: WAGAKRLVLRRE;Hnilova et al, 2008;AuBP2: WALRRSIRRQSY;Hnilova et al, 2008; Cys-BP: CKPKEL-PEKLPELKPK(Ahx-Ahx-Biotin)C-NH2; Zernia et al, 2016), stainless steel (e.g., MS-S1: ATIHDAFYSAPE; Zuo, Örnek, & Wood, 2005; MS-S2: NLNPNTASAMHV; Zuo et al, 2005;MTWDPSLASPRS;Vreuls et al, 2010), silicon (e.g., TBP-1: RKLPDAPGMHTW; Sano, Sasaki, & Shiba, 2005; P2:LLADTTHHRPWT; Estephan et al, 2011;Ramakrishnan, Martin, Cloitre, Firlej, & Gergely, 2014;P3: SPGLSLVSHMQT;Estephan et al, 2011;Ramakrishnan et al, 2014;PC4: SLVSHMQT;Ramakrishnan et al, 2015), polystyrene (PS; e.g., PB-TUB: VHWDFRQWWQPS ;Qiang et al, 2017;PS-19: RAFIASRRIKRP;Kumada et al, 2006;c02: YLTMPTP;Serizawa, Techawanitchai, & Matsuno, 2007; Tachystatin A2 (TA2): YSRCQLQGFNCVVRSYGLPTIPCCRGLTCRSYFPGSTYGRCQRY; Rübsam, Weber, Jakob, & Schwaneberg, 2018), and polypropylene (PP; e.g., TSDIKSRSPHHR, HTQNMRMYEPWF; Cunningham, Lowe, O'brien, Wang, & Wilkins, 2011; Cecropin A (CecA): KWKLFKKIEKVGQNIRD-GIIKAGPAVAVVGQATQIAK; Rübsam, Stomps, Böker, Jakob, & Schwaneberg, 2017; liquid chromatography peak I (LCI): AIKLVQSPNGN-FAASFVLDGTKWIFKSKYYDSSKGYWVGIYEVWDRK; Rübsam et al, 2017).…”

“…Here, we report a universal immobilization platform based on adhesion-promoting peptides (Matter-tags). Three adhesion promoting peptides, CecA, LCI, and TA2 were selected due to their reported binding to synthetic polymers (PS, PP, PUR, triblock copolymers like PIB 1000 -PEG 6000 -PIB 1000 and PMOXA 15 -PDMS 68 -PMOXA 15 ; Islam, Apitius, Jakob, & Schwaneberg, 2019;Klermund, Poschenrieder, & Castiglione, 2016;Noor et al, 2012;Rübsam et al, 2017;Rübsam, Weber et al, 2018;Rübsam, Davari, Jakob, & Schwaneberg, 2018), and natural surfaces, for example, plant leaves (Meurer et al, 2017;Schwinges et al, 2019). The developed immobilization platform enables to cover a broad spectrum of industrially and medically relevant materials.…”

Ultrahigh‐throughput screening system for directed polymer binding peptide evolution

“…All constructs were cloned according to the protocols published before (Figure a–c; Rubsam et al, ). The synthetic gene ompA‐lci‐17xhelix‐e‐eptitope‐estA* (*indicates inactivated EstA by introduction of the mutation S38A; S. Becker et al, ) was cloned in a pET22b(+) backbone applying “phosphorothioate‐based ligase‐independent gene cloning” (PLICing; Blanusa, Sadeghi, & Schwaneberg, ).…”

“…LCI was used, for example, as adhesion promotor for directed CueO laccase evolution enabling semipurification in 96‐well microtiter plates (MTPs; Zou, Rubsam, & Schwaneberg, ). The peptide was furthermore optimized in a directed peptide evolution campaign (PeP evo , epPCR based) for strengthened PP‐binding in the presence of Triton X‐100 (Rubsam, Jakob, & Schwaneberg, ). Additionally, LCI was engineered in a knowledge gaining directed evolution (KnowVolution) campaign reporting a 5.4‐fold improved PP‐binder (Y29R and G35R) in the presence of Triton X‐100 (Rübsam, Jakob, & Schwaneberg, ).…”